- Email: [email protected]
Effect of preanalytical variables on myeloperoxidase levels
Clinica Chimica Acta 411 (2010) 1650–1655
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Effect of preanalytical variables on myeloperoxidase levels Andrea Elisabet Wendland a,d,⁎, Joíza Lins Camargo b, Carisi Anne Polanczyk c,d a
Central Public Health Laboratory, Porto Alegre, Brazil Department of Clinical Pathology, Hospital de Clínicas de Porto Alegre, Brazil c Department of Cardiology, Hospital de Clínicas de Porto Alegre, Brazil d Cardiology Graduate Program, Federal University of Rio Grande do Sul, Brazil b
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 14 June 2010 Accepted 14 June 2010 Available online 22 June 2010 Keywords: Myeloperoxidase Inflammation Cardiovascular disease Preanalytical variables
a b s t r a c t Background: Myeloperoxidase (MPO) levels have prognostic value in cardiovascular events, but information about preanalytical variables is scarce. This study evaluated the effect of different sample types and storage conditions on MPO measurements. Methods: Plasma and serum samples [heparinized plasma (MPO-Hep), EDTA plasma (MPO-EDTA), and serum with (MPO-Gel) and without separator gel (MPO-Serum)] from 40 volunteers were assayed for MPO by ELISA (Bioxytech® MPO-EIA™ kit). To evaluate MPO stability, samples were stored at 18–25 °C, at 2–8 °C and at − 20 °C and − 80 °C for predetermined periods. Results: MPO levels ranged from 16 to 69 ng/ml and were higher in patients with heart disease compared to healthy volunteers (35.0 vs. 24.9 ng/ml; P = 0.03). There were no statistical differences between MPO-Hep, MPO-Gel and MPO-Serum (P N 0.05), and MPO-Hep showed a good correlation with MPO-Gel and MPOSerum (r = 0.775 and r = 0.792; P b 0.001). No correlation between MPO-Hep and MPO-EDTA was found (r = 0.21; P = 0.20), and mean MPO-EDTA value was 1.8 times higher than MPO-Hep (51.4 vs. 28.7 ng/ml; P b 0.001). Mean differences (ng/ml) between MPO-Hep and MPO-EDTA, MPO-Gel and MPO-Serum were 24.7 (19.5–30.0), 0.225 (− 2.54–2.99), and 1.55 (− 1.16–4.26), respectively. Conclusions: EDTA had a significant effect on MPO results. MPO-Hep values correlate well with MPO-Gel and MPO-Serum. MPO levels seem to be stably frozen at − 20 °C or − 80 °C over 6 months depending on preanalytical handling. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Inflammation plays an important role in all stages of atherosclerotic plaque development, from initial dysfunction to formation of atheroma, plaque rupture and the resulting thrombotic complications [1–3]. Different inflammatory biomarkers have been studied [4]. Rupture and erosion of plaques with formation of an intramural thrombus are the most important morphological changes in the transformation of stable coronary lesions into clinically unstable ones. Lymphocytes and monocytes seem to make an important contribution to the pathophysiology of cardiovascular disease, in particular through the generation of proinflammatory cytokines. However, polymorphonuclear neutrophils (PMNs) may also be involved, and may modulate and signal inflammatory pathways by secreting enzymes, such as myeloperoxidase, that interact with target organs [5].
Abbreviations: PMNs, polymorphonuclear neutrophils; MPO, myeloperoxidase; NO, nitric oxide; CRP, C-reactive protein; hs, high sensitivity; NT-proBNP, N-terminal pro-Btype natriuretic peptide. ⁎ Corresponding author. Av. Prof. Cristiano Fischer, 2062/526 91410-000, Porto Alegre, RS, Brazil. Tel.: + 55 51 3339 2796. E-mail address: [email protected] (A.E. Wendland). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.06.015
Myeloperoxidase (MPO), the most abundant component of azurophilic granules of leukocytes, is released after activation and degranulation of leukocytes, contributes to innate immune defenses, and has been shown to participate in the promotion and propagation of atherosclerosis [3]. MPO generates free radicals and diffusible oxidants, and can, therefore, oxidize LDL cholesterol and promote uptake by macrophages and foam cell formation [6,7]. It may also cause oxidative changes in HDL and impair reverse transport of cholesterol [8]. MPO activates metalloproteinases and promotes destabilization and rupture of the atherosclerotic plaque [9,10]; it catalytically consumes nitric oxide (NO) derived from endothelium, reduces its bioavailability and affects its vasodilator and antiinflammatory functions [11,12]; and may also contribute to adverse ventricular remodeling after infarction [12,13]. Several studies have demonstrated the prognostic association of high levels of MPO and adverse cardiovascular events in healthy individuals [14], patients with chronic heart disease [15] or acute heart disease [16–18], patients being examined because of chest pain [19], and patients with heart failure [20,21]. However, its clinical use depends on further studies to define accurate and reproducible analytical methods. Moreover, preanalytical factors, such as sample type, handling and storage, must be fully evaluated [22]. The preanalytical sources of MPO
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
variability are still unknown, but the quality of assay results and correct clinical management depend on their accurate definition. This study evaluated the effect of different sample types and storage conditions on MPO measurements. 2. Materials and methods 2.1. Samples The Clinical Laboratory Standard Institute (CLSI) protocol was used for method validation (CLSI EP9-A Protocol: Method comparison and bias estimation using patient samples. Approved Guideline, 1995. Clinical Laboratory Standard Institute, Wayne. PA). Blood samples were collected from 40 volunteers (17 men) aged 21 to 75 years. Twenty-five participants were healthy and 15 had cardiac disease. Eight patients had stable coronary artery disease, 3 had acute coronary syndrome, and 4 had heart failure. All participants answered a questionnaire about clinical information and signed an informed consent form. This study was conducted at the Hospital de Clínicas de Porto Alegre, RS, Brazil, and was approved by its Committee on Research Ethics. 2.2. Blood collection and sample storage The study design is illustrated schematically in Fig. 1. Four blood samples were obtained from each study participant (Vacuette® tubes Greiner Bio-one), and one specimen was prepared from each sample: heparinized plasma (coated tube with sodium heparin; MPO-Hep), EDTA plasma (coated tube with EDTAK3; MPO-EDTA), serum (coated tube with micronized silica particles, to activate clotting; MPOSerum), and serum with separator gel (coated tube with micronized silica particles and a barrier gel; MPO-Gel). Immediately after sample collection, tubes were mixed gently to homogenize the material and were immediately kept on ice bath for no longer than 30 min until centrifugation at 2000 g for 10 min. After centrifugation, serum (with and without gel) and plasma (heparinized and with EDTA) specimens were divided into aliquots to be used in the study procedures. To compare MPO results for the different tube preparations, the following specimens were taken from the samples from each volunteer: aliquots of plasma were obtained from the tubes with heparin and EDTA, and aliquots of serum, from tubes with and without gel. All these specimens were stored in a freezer at −20 °C for 36 h until measurement.
1651
To evaluate the stability of MPO according to different storage periods and temperatures, aliquots of heparinized plasma, which is the sample of choice according to the manufacturer of the kit, were separated (n = 40) and stored at room temperature (18–25 °C) for 3 days; in a refrigerator (2–8 °C) for 3 and 7 days; in a freezer at −20 °C for 7, 30, 90 and 180 days and in a freezer at − 80 °C for 30, 90 and 180 days. The measurement at 36 h after collection was considered the baseline for this stability study. The choice of this point was based on bench workflow feasibility and the manufacturer's instructions. For the analytical imprecision studies, 20 aliquots of MPO-Hep were obtained from a single sample stored at −20 °C and measured on the same day (intraassay CV) and 20 aliquots from a single sample were stored at − 80 °C and measured on different days (interassay CV) throughout the assaying period, 2.3. Sample measurement and methodology MPO level was measured using the ELISA method with a Bioxytech® MPO-EIA™ kit (OXIS Health Products, Inc. USA). A standard MPO solution (50 ng/ml) was poured on each plate to plot a 6-point standard curve by serial dilutions. All samples were diluted at a 1:10 ratio, with the sample diluting buffer, according to the manufacturer's instructions, so that all concentrations were within the method's range of linearity (1.56–25 ng/ml). Absorption was read at 405 nm using an ETI-Max 3000 DiaSorin analyzer. 2.4. Statistical analysis Continuous variables are presented as mean ± SD, and categorical variables, as absolute numbers and percentages. The MPO levels had a normal distribution. Differences between means were evaluated using the Student t test for continuous variables and the χ2 for categorical variables. Correlations between sample types were evaluated using the Pearson correlation coefficient, and mean differences were analyzed using the Bland–Altman method. Sample stability under different storage conditions was evaluated using repeated-measures ANOVA and intraclass correlation coefficients. Data were analyzed using the statistical program SPSS 14.0, and differences were classified as statistically significant at P b 0.05. 3. Results Characteristics of individuals participating in the study are shown in Table 1. Mean age was 45 (21–75) years. MPO levels ranged from 16 to 69 ng/ml (mean: 28.7 ng/ml), with no difference between men and
Fig. 1. Study design. FICF: free and informed consent form; RT: room temperature; CV: coefficient of variation; EDTA: ethylenediaminetetraacetic acid.
1652
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
Table 1 Characteristics of individuals participating in the study. Individuals (n = 40) Age, years Sex Males Females Individuals Controls With cardiac disease Medication AAS Statins Heparin Ticlopidine Angina Dyslipidemia Diabetes mellitus Arterial hypertension Renal insufficiency Inflammatory disease Neoplasm Myeloperoxidase (ng/ml)
44.7 (21–75) 17 (42.5%) 23 (57.5%) 25 (62.5%) 15 (37.5%) 11 (27.5%) 11 (27.5%) 6 (15%) 1 (2.5%) 4 (10%) 13 (32.5%) 3 (7.5%) 13 (32.5%) 4 (10%) 2 (5%) 1 (2.5%) 28.7 (16–69)
Age and myeloperoxidase: mean (minimum and maximum); AAS: acetyl salicylic acid.
women (31.0±12.4 vs. 27.0±12.2 ng/ml; P=0.31). MPO levels in the group of patients with cardiac disease were significantly higher than those in the group of healthy volunteers (35.0±15.4 vs. 24.9±8.2 ng/ml; P=0.03). The analytical intraassay and interassay CVs were 8.1% and 10.1%.
3.1. Effect of anticoagulants The comparison of MPO levels in different sample types revealed no significant differences between MPO-Hep (28.7±12.3 ng/ml) and serum collected in tubes with gel (28.9±13.4 ng/ml; P=0.87) or serum collected in tubes without gel (30.2±13.7 ng/ml; P=0.25). However, the results of samples collected in tubes containing EDTA for anticoagulation were significantly different from the results found for the MPO-Hep samples, with mean results about 1.8 times greater (51.4±15.5 vs. 28.7±12.3 ng/ml; Pb 0.001) (Fig. 2). The correlations between the results found for MPO-Gel and MPO-Serum and those for MPO-Hep were good (r=0.775 and r=0.792; Pb 0.001). There was no correlation between MPO-EDTA and MPO-Hep (r = 0.21; P = 0.20) (Fig. 3). The Bland–Altman plots [23] revealed that the mean differences of MPO-Hep from MPO-EDTA, MPO-Gel and MPO-Serum were 24.7 ng/ml (95%CI 19.5–30.0 ng/ml), 0.225 ng/ml (95%CI −2.54–2.99 ng/ml) and 1.55 ng/ml (95%CI −1.16–4.26 ng/ml) (Fig. 4).
Fig. 3. MPO levels in different sample types: correlation between MPO heparin and MPO-EDTA (a), MPO gel (b) and MPO serum (c). The dotted lines represent the line of equality at which Y = X.
3.2. Effect of storage conditions
Fig. 2. Effect of the different anticoagulants on MPO levels: comparison between plasma (with heparin and EDTA) and serum (with and without separator gel).
MPO levels for samples stored at different temperatures and for different periods of time are illustrated in Fig. 5. An increase in MPO levels in stored samples compared to the levels found in the baseline assay (36 h), (28.7±12.3 vs. 28.9±15.5 and 29.6± 15.2 ng/ml; PN 0.05, for baseline, 3 days at RT and refrigerator) being significant only from the 7day storage period, both for samples stored in the refrigerator (2–8 °C) and those stored at −20 °C (28.7 ±12.3 vs. 37.1±16.7 ng/ml; Pb 0.001 and 33.3±15.9 ng/ml; P=0.006) was observed. This increase remained constant for all other aliquots stored at all remaining time points. There were no differences between MPO levels of samples stored for 30, 90 or 180 days at −20 °C (37.9± 17.2 vs. 36.0 ±18.3 ng/ml; P =0.12 vs. 42.0 ±23.6 ng/ml; P= 0.21) or at −80 °C (36.7± 16.6 vs. 35.3 ± 19.0 ng/ml; P= 0.33 vs. 42.3±28.6 ng/ml; P=0.16). The analysis of sample stability over the entire storage time revealed a moderate
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
1653
Fig. 5. MPO levels assayed during storage. An increase in MPO levels was observed in stored aliquots along time, more markedly from 7 days onwards; increase then remained constant at later time points.
Fig. 4. Bland–Altman difference plots for MPO levels in different sample types: the solid line represents the mean difference between the reference sample (heparin) and EDTA (a), serum with (b) and without separator gel (c).
correlation between results (intraclass correlation coefficient [ICC]: 0.598; 95% CI 0.480–0.724; Pb 0.001). The comparison of samples stored at the same temperatures over time revealed better correlations: for those stored at −20 °C, ICC was 0.678 (95% CI 0.540–0.798; Pb 0.001), and for those stored at −80 °C, ICC was 0.667 (95% CI 0.507–0.795; Pb 0.001).
4. Discussion This study compared MPO results obtained from the analysis of heparinized plasma, which is the sample of choice according to the kit's manufacturer, with results obtained from plasma with EDTA and from serum collected in tubes with or without separator gel. No differences were found between MPO results of MPO-Hep and serum in tubes with or without separator gel. However, a significant difference was found in the MPO-EDTA results. Mean results of MPO-EDTA were about 107% greater than the results of MPO-Hep, and nine of the samples had results that were more than 150% greater.
Current knowledge suggests that MPO, a potential cause of progression and instability of atherosclerotic lesions during acute ischemic events, may be used as a marker of cardiovascular disease and provide information to improve patient diagnosis and prognosis [24]. However, little is known about the effect of preanalytical factors on MPO measurements. Studies have used different cut-off points because of differences in sample types and handling procedures. Therefore, the limits within which results can be used as the basis for decisions are uncertain, and further studies should evaluate analytical and preanalytical performances [22]. Several studies that evaluated other markers of cardiac disease, as well as the only two other studies about MPO published to date, found that preanalytical factors may affect measurements [25–35]. Chang et al. [31] compared MPO results from heparinized plasma and serum samples and found that serum results were considerably higher, although this could be attributed to the time the sample remained at room temperature for clot formation before centrifugation and separation of the material, and to the fact that the enzyme apparently continued to release from the leukocytes when whole blood was left standing at room temperature. No increases in MPO concentrations were found in samples place on ice bath after collection. Our study did not detect this difference: all our samples were put in an ice bath immediately after collection, and enzyme release was avoided. All tubes used to collect serum contained a clot activator. Samples collected in different tubes were found to have higher levels of MPO in serum and heparinized plasma than in plasma with EDTA or citrate [32]. In the heparinized plasma samples, MPO levels were about 10% higher than in EDTA plasma, and values for the serum samples were up to 100% higher. There was no significant difference between EDTA and citrate plasma in that study. Those findings differ from our results, in which values were considerably higher in EDTA samples. Some methodological and sample handling differences may explain the differences observed in the two studies. While in that study an automated chemiluminescent microparticle immunoassay was used, we used a manual method based on a sandwich ELISA and analyzed a larger number of samples. In our study samples were place on ice bath immediately after the collection. The changes observed in EDTA samples may be explained by the fact that when blood is collected in EDTA, neutrophils undergo morphological changes that are dependent on contact time and concentration, and minor changes may appear even at an optimal anticoagulant concentration. These changes include swelling of the neutrophils with loss of lobe structure, followed by loss of granulation and the appearance of vacuoles in the cytoplasm and in the nucleus of the cells. Three hours after blood collection, cells can disintegrate [36]. This loss of neutrophil granulation may be a contributing factor to the increase of MPO concentrations in plasma collected with EDTA. To avoid incorrect clinical
1654
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
evaluations or risk stratification, blood collection in tubes with EDTA should be further investigated, and reference values should be determined. Similar discrepancies have been described for other markers. Roberts et al. [33] compared hs-CRP measurements in serum, heparinized plasma and EDTA plasma and found that, in spite of excellent correlations, EDTA plasma samples had lower levels. In contrast, another study found no significant differences between samples of serum, heparinized plasma and EDTA plasma collected simultaneously [34]. Our study also evaluated the stability of MPO according to different storage times and temperatures, from 3 days at room temperature (25 °C) or in a refrigerator (2–8 °C), to 6 months in a freezer (−20 °C and −80 °C). There was an increase in MPO levels from the first measurement to all others, and this increase was more marked from 7 days onwards. However, this difference does not seem to be a function of storage time or temperature because no systematic elevation of levels was found over time in the samples stored for longer periods (30, 90 and 180 days) at −20 °C or at −80 °C. Furthermore, aliquots to determine the analytical CVs also failed to show an increase in MPO levels at longer storage times. This difference, in agreement with the findings reported by Chang et al. [31], may be explained by the time that the plasma was in contact with cellular constituents while the samples remained at room temperature, which was approximately 2 h, from centrifugation to separation of the aliquots. In the study conducted by Chang et al. [31], the time during which the whole blood samples remained at room temperature before undergoing centrifugation had a significant effect on MPO levels in plasma; moreover, that increase was proportional to time, and the concentration was almost double when samples remained at room temperature for 1 h before centrifugation. No increases in MPO concentrations were found in samples put in an ice bath after collection, not even when centrifugation was performed at room temperature. In contrast, leukocytes apparently continued to release MPO in the samples stored at room temperature up to the time that they underwent centrifugation. Results reported by Shih et al. [32] also support this hypothesis. Plasma samples had a b10% difference in MPO levels after supernatant separation, storage at room temperature for 2 days, storage at 2–8 °C for 8 days and after 3 freeze–thaw cycles. However, the same study found a 4 times increase in the MPO levels of samples collected in tubes containing heparin and stored at room temperature for 2 h before centrifugation, whereas samples kept in an ice bath remained relatively stable. In contrast with the studies conducted by Chang et al. [31] and Shih et al. [32], who found that MPO levels increased in whole blood before sample centrifugation, we found that enzyme release seems to take place even after centrifugation. Therefore, even when samples are placed in an ice bath after collection, plasma must be separated immediately after centrifugation to avoid false increases in levels resulting from the continuous enzyme release into the plasma that is in contact with the cells. Our study results and those reported by Shih et al. [32] suggest that once plasma has been separated from the cellular constituents, MPO concentration is not apparently affected by storage temperature or time, which is useful information for investigators who intend to store samples for future measurements. What is of fundamental importance in MPO measurements and significantly affects the results is the time during which the sample, as whole blood or as plasma after centrifugation, remains in contact with cellular constituents. A common practice in clinical laboratories is to draw blood in serum-separator tubes because of the advantage of the barrier gel, which facilitates rapid separation of serum from cell constituents of blood and prevents hemolysis during prolonged storage. However, the measured concentrations of certain analytes may be reduced if specimens are stored in this type of tube because of adsorption by the
gel [35]. Our study did not show any significant differences between samples collected with heparin and serum with or without separator gel. Furthermore, despite some pair discrepancies, all of them within the expected total error for this analyte [37], there was a good correlation between MPO results for these 3 types of sample. This suggests that blood for the analysis of several cardiac markers may be collected in the same tube, which may shorten collection time, reduce costs and increase patient comfort. Some limitations to our study should be acknowledged. The fact that we did not know that the enzyme was likely to be released from the leukocytes into plasma after samples have been centrifuged, even when samples were put in an ice bath soon after collection, made our results difficult to interpret. Although they indicated the possibility that storage did not affect MPO levels, confirmation was only obtained from studies published subsequently. Studies in which samples remain in contact with cells before aliquot separation at room temperature and in an ice bath for predetermined times should be conducted to further explain the effect of this factor and define the rate at which MPO levels change. We evaluated the effect of storage on heparinized plasma samples only, and further studies should investigate such effect on other sample types. This study demonstrated that the collection of MPO in tubes containing EDTA alters in expressive way the results. Other aspects as time of storage and type of sample had no effect in the results. Further studies should be conducted to evaluate analytical and preanalytical variability. Measurement units should be standardized, populationbased reference ranges should be determined, and standard, automated and commercially available tests should be developed to shorten test time, facilitate use, and reduce analytical imprecision, which are the basic benchmarks to move this marker from the research environment to routine clinical practice.
References [1] Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135–43. [2] Hansson GK. Mechanisms of disease: inflammation, atherosclerosis and coronary artery disease. N Engl J Med 2005;352:1685–95. [3] Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol 2005;25:1102–11. [4] Apple FS, Wu AHB, Mair J, Ravkilde J, Panteghini M, Tate J, et al. Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem 2005;51:810–24. [5] Naruko T, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, et al. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation 2002;106:2894–900. [6] Podrez EA, Schmitt D, Hoff HF, Hazen SL. Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro. J Clin Invest 1999;103:1547–60. [7] Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda SE, et al. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 2002;277:46116–22. [8] Nicholls S, Zheng L, Hazen SL. Formation of dysfunctional high-density lipoprotein by myeloperoxidase. Trends Cardiovasc Med 2005;15:212–9. [9] Hazen SL. Myeloperoxidase and plaque vulnerability. Arterioscler Thromb Vasc Biol 2004;24:1143–6. [10] Sugiyama S, Kugiyama K, Aikawa M, Nakamura S, Ogawa H, Libby P. Hypochlorous acid, a macrophage product, induces endothelial apoptosis and tissue factor expression. Involvement of myeloperoxidase-mediated oxidant in plaque erosion and thrombogenesis. Arterioscler Thromb Vasc Biol 2004;24:1309–14. [11] Vita JA, Brennan ML, Gokce N, Mann SA, Goormastic M, Shishehbor MH, et al. Serum myeloperoxidase levels independently predict endothelial dysfunction in humans. Circulation 2004;110:1134–9. [12] Baldus S, Heitzer T, Eiserich JP, Lau D, Mollnau H, Ortak M, et al. Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion. Free Radic Biol Med 2004;37:902–11. [13] Vasilyev N, Williams T, Brennan ML, Unzek S, Zhou X, Heinecke JW, et al. Myeloperoxidase-generated oxidants modulate left ventricular remodeling but not infarct size after myocardial infarction. Circulation 2005;112:2812–20. [14] Meuwese MC, Stroes WS, Hazen SL, van Miert JN, Kuivenhoven JA, Schaub RG, et al. Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals. J Am Coll Cardiol 2007;50:159–65. [15] Stefanescu A, Braun S, Ndrepepa G, Koppara T, Pavaci H, Mehilli J, et al. Prognostic value of plasma myeloperoxidase concentration in patients with stable coronary artery disease. Am Heart J 2008;155:356–60.
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655 [16] Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Münzel T, et al. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation 2003;108:1440–5. [17] Cavusoglu E, Ruwende C, Eng C, Chopra V, Yanamadala S, Clark LT, et al. Usefulness of baseline plasma myeloperoxidase levels as an independent predictor of myocardial infarction at two years in patients presenting with acute coronary syndrome. Am J Cardiol 2007;99:1364–8. [18] Mocatta TJ, Pilbrow AP, Cameron VA, Senthilmohan R, Frampton CM, Richards AM, et al. Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction. J Am Coll Cardiol 2007;49:1993–2000. [19] Brennan ML, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles RJ, et al. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med 2003;349:1595–604. [20] Tang WHW, Brennan ML, Philip K, Tong W, Mann S, Van Lente F, et al. Plasma myeloperoxidase levels in patients with chronic heart failure. Am J Cardiol 2006;98: 796–9. [21] Tang WHW, Tong W, Troughton RW, Martin MG, Shrestha K, Borowski A, et al. Prognostic value and echocardiographic determinants of plasma myeloperoxidase levels in chronic heart failure. J Am Coll Cardiol 2007;49:2364–70. [22] Morrow DA. Appraisal of myeloperoxidase for evaluation of patients with suspected acute coronary syndromes. J Am Coll Cardiol 2007;49:2001–2. [23] Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;8:307–10. [24] Roman RM, Wendland AE, Polanczyk CA. Myeloperoxidase and coronary arterial disease: from research to clinical practice. Arq Bras Cardiol 2007;91:e12–9. [25] Stiegler H, Fischer Y, Jimenez JFV, Graf J, Filzmaier K, Fausten B, et al. Lower cardiac troponin T and I results in heparin-plasma than in serum. Clin Chem 2000;46: 1338–44. [26] Gerhardt W, Nordin G, Herbert AK, Burzell BL, Isaksson A, Gustavsson E, et al. Troponin T and I assays show decreased concentrations in heparin plasma compared
[27]
[28] [29] [30]
[31]
[32]
[33] [34] [35] [36] [37]
1655
with serum: lower recoveries in early than in late phases of myocardial injury. Clin Chem 2000;46:817–21. Ledue TB, Rifai N. Preanalytic and analytic sources of variation in C-reactive protein measurement: implications for cardiovascular disease risk assessment. Clin Chem 2003;49:1258–71. Lippi G, Salvagno GL, Montagnana M, Guidi GC. Measurement of Elecsys NTproBNP in serum, K2 EDTA and heparin plasma. Clin Biochem 2007;40:747–8. Shimizu H, Aono K, Masuta K, Asada H, Misaki A, Teraoka H. Stability of brain natriuretic peptide (BNP) in human blood samples. Clin Chim Acta 1999;285:169–72. Nowatzke WL, Cole TG. Stability of N-terminal pro-brain natriuretic peptide after storage frozen for one year and after multiple freeze–thaw cycles. Clin Chem 2003;49:1560–2. Chang PY, Wu TL, Hung CC, Tsao KC, Sun CF, Wu LL, et al. Development of an ELISA for myeloperoxidase on microplate: normal reference values and effect of temperature on specimen preparation. Clin Chim Acta 2006;373:158–63. Shih J, Datwyler SA, Hsu SC, Matias MS, Pacenti DP, Lueders C, et al. Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations. Clin Chem 2008;54:1076–9. Roberts WL, Schwarz EL, Ayanian S, Rifai N. Performance characteristics of a point of care C-reactive protein assay. Clin Chim Acta 2001;314:255–9. Rothkrantz-Kos S, Schmitz PJ, Bekers O, Menheere PPCA, van Dieijen-Visser MP. High-sensitivity C-reactive protein methods examined. Clin Chem 2002;48:359–62. Dasgupta A, Chow L, Tso G, Nazareno L. Stability of NT-proBNP in serum specimens collected in Becton Dickinson Vacutainer (SST) tubes. Clin Chem 2003;49:958–60. Narayanan S. The preanalytical phase — an important component of laboratory medicine. Am J Clin Pathol 2000;113:429–52. Biological Variation Database specifications — Westgard QC http://www.westgard. com/biodatabase1.htm.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Effect of preanalytical variables on myeloperoxidase levels Andrea Elisabet Wendland a,d,⁎, Joíza Lins Camargo b, Carisi Anne Polanczyk c,d a
Central Public Health Laboratory, Porto Alegre, Brazil Department of Clinical Pathology, Hospital de Clínicas de Porto Alegre, Brazil c Department of Cardiology, Hospital de Clínicas de Porto Alegre, Brazil d Cardiology Graduate Program, Federal University of Rio Grande do Sul, Brazil b
a r t i c l e
i n f o
Article history: Received 15 January 2010 Received in revised form 14 June 2010 Accepted 14 June 2010 Available online 22 June 2010 Keywords: Myeloperoxidase Inflammation Cardiovascular disease Preanalytical variables
a b s t r a c t Background: Myeloperoxidase (MPO) levels have prognostic value in cardiovascular events, but information about preanalytical variables is scarce. This study evaluated the effect of different sample types and storage conditions on MPO measurements. Methods: Plasma and serum samples [heparinized plasma (MPO-Hep), EDTA plasma (MPO-EDTA), and serum with (MPO-Gel) and without separator gel (MPO-Serum)] from 40 volunteers were assayed for MPO by ELISA (Bioxytech® MPO-EIA™ kit). To evaluate MPO stability, samples were stored at 18–25 °C, at 2–8 °C and at − 20 °C and − 80 °C for predetermined periods. Results: MPO levels ranged from 16 to 69 ng/ml and were higher in patients with heart disease compared to healthy volunteers (35.0 vs. 24.9 ng/ml; P = 0.03). There were no statistical differences between MPO-Hep, MPO-Gel and MPO-Serum (P N 0.05), and MPO-Hep showed a good correlation with MPO-Gel and MPOSerum (r = 0.775 and r = 0.792; P b 0.001). No correlation between MPO-Hep and MPO-EDTA was found (r = 0.21; P = 0.20), and mean MPO-EDTA value was 1.8 times higher than MPO-Hep (51.4 vs. 28.7 ng/ml; P b 0.001). Mean differences (ng/ml) between MPO-Hep and MPO-EDTA, MPO-Gel and MPO-Serum were 24.7 (19.5–30.0), 0.225 (− 2.54–2.99), and 1.55 (− 1.16–4.26), respectively. Conclusions: EDTA had a significant effect on MPO results. MPO-Hep values correlate well with MPO-Gel and MPO-Serum. MPO levels seem to be stably frozen at − 20 °C or − 80 °C over 6 months depending on preanalytical handling. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Inflammation plays an important role in all stages of atherosclerotic plaque development, from initial dysfunction to formation of atheroma, plaque rupture and the resulting thrombotic complications [1–3]. Different inflammatory biomarkers have been studied [4]. Rupture and erosion of plaques with formation of an intramural thrombus are the most important morphological changes in the transformation of stable coronary lesions into clinically unstable ones. Lymphocytes and monocytes seem to make an important contribution to the pathophysiology of cardiovascular disease, in particular through the generation of proinflammatory cytokines. However, polymorphonuclear neutrophils (PMNs) may also be involved, and may modulate and signal inflammatory pathways by secreting enzymes, such as myeloperoxidase, that interact with target organs [5].
Abbreviations: PMNs, polymorphonuclear neutrophils; MPO, myeloperoxidase; NO, nitric oxide; CRP, C-reactive protein; hs, high sensitivity; NT-proBNP, N-terminal pro-Btype natriuretic peptide. ⁎ Corresponding author. Av. Prof. Cristiano Fischer, 2062/526 91410-000, Porto Alegre, RS, Brazil. Tel.: + 55 51 3339 2796. E-mail address: [email protected] (A.E. Wendland). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.06.015
Myeloperoxidase (MPO), the most abundant component of azurophilic granules of leukocytes, is released after activation and degranulation of leukocytes, contributes to innate immune defenses, and has been shown to participate in the promotion and propagation of atherosclerosis [3]. MPO generates free radicals and diffusible oxidants, and can, therefore, oxidize LDL cholesterol and promote uptake by macrophages and foam cell formation [6,7]. It may also cause oxidative changes in HDL and impair reverse transport of cholesterol [8]. MPO activates metalloproteinases and promotes destabilization and rupture of the atherosclerotic plaque [9,10]; it catalytically consumes nitric oxide (NO) derived from endothelium, reduces its bioavailability and affects its vasodilator and antiinflammatory functions [11,12]; and may also contribute to adverse ventricular remodeling after infarction [12,13]. Several studies have demonstrated the prognostic association of high levels of MPO and adverse cardiovascular events in healthy individuals [14], patients with chronic heart disease [15] or acute heart disease [16–18], patients being examined because of chest pain [19], and patients with heart failure [20,21]. However, its clinical use depends on further studies to define accurate and reproducible analytical methods. Moreover, preanalytical factors, such as sample type, handling and storage, must be fully evaluated [22]. The preanalytical sources of MPO
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
variability are still unknown, but the quality of assay results and correct clinical management depend on their accurate definition. This study evaluated the effect of different sample types and storage conditions on MPO measurements. 2. Materials and methods 2.1. Samples The Clinical Laboratory Standard Institute (CLSI) protocol was used for method validation (CLSI EP9-A Protocol: Method comparison and bias estimation using patient samples. Approved Guideline, 1995. Clinical Laboratory Standard Institute, Wayne. PA). Blood samples were collected from 40 volunteers (17 men) aged 21 to 75 years. Twenty-five participants were healthy and 15 had cardiac disease. Eight patients had stable coronary artery disease, 3 had acute coronary syndrome, and 4 had heart failure. All participants answered a questionnaire about clinical information and signed an informed consent form. This study was conducted at the Hospital de Clínicas de Porto Alegre, RS, Brazil, and was approved by its Committee on Research Ethics. 2.2. Blood collection and sample storage The study design is illustrated schematically in Fig. 1. Four blood samples were obtained from each study participant (Vacuette® tubes Greiner Bio-one), and one specimen was prepared from each sample: heparinized plasma (coated tube with sodium heparin; MPO-Hep), EDTA plasma (coated tube with EDTAK3; MPO-EDTA), serum (coated tube with micronized silica particles, to activate clotting; MPOSerum), and serum with separator gel (coated tube with micronized silica particles and a barrier gel; MPO-Gel). Immediately after sample collection, tubes were mixed gently to homogenize the material and were immediately kept on ice bath for no longer than 30 min until centrifugation at 2000 g for 10 min. After centrifugation, serum (with and without gel) and plasma (heparinized and with EDTA) specimens were divided into aliquots to be used in the study procedures. To compare MPO results for the different tube preparations, the following specimens were taken from the samples from each volunteer: aliquots of plasma were obtained from the tubes with heparin and EDTA, and aliquots of serum, from tubes with and without gel. All these specimens were stored in a freezer at −20 °C for 36 h until measurement.
1651
To evaluate the stability of MPO according to different storage periods and temperatures, aliquots of heparinized plasma, which is the sample of choice according to the manufacturer of the kit, were separated (n = 40) and stored at room temperature (18–25 °C) for 3 days; in a refrigerator (2–8 °C) for 3 and 7 days; in a freezer at −20 °C for 7, 30, 90 and 180 days and in a freezer at − 80 °C for 30, 90 and 180 days. The measurement at 36 h after collection was considered the baseline for this stability study. The choice of this point was based on bench workflow feasibility and the manufacturer's instructions. For the analytical imprecision studies, 20 aliquots of MPO-Hep were obtained from a single sample stored at −20 °C and measured on the same day (intraassay CV) and 20 aliquots from a single sample were stored at − 80 °C and measured on different days (interassay CV) throughout the assaying period, 2.3. Sample measurement and methodology MPO level was measured using the ELISA method with a Bioxytech® MPO-EIA™ kit (OXIS Health Products, Inc. USA). A standard MPO solution (50 ng/ml) was poured on each plate to plot a 6-point standard curve by serial dilutions. All samples were diluted at a 1:10 ratio, with the sample diluting buffer, according to the manufacturer's instructions, so that all concentrations were within the method's range of linearity (1.56–25 ng/ml). Absorption was read at 405 nm using an ETI-Max 3000 DiaSorin analyzer. 2.4. Statistical analysis Continuous variables are presented as mean ± SD, and categorical variables, as absolute numbers and percentages. The MPO levels had a normal distribution. Differences between means were evaluated using the Student t test for continuous variables and the χ2 for categorical variables. Correlations between sample types were evaluated using the Pearson correlation coefficient, and mean differences were analyzed using the Bland–Altman method. Sample stability under different storage conditions was evaluated using repeated-measures ANOVA and intraclass correlation coefficients. Data were analyzed using the statistical program SPSS 14.0, and differences were classified as statistically significant at P b 0.05. 3. Results Characteristics of individuals participating in the study are shown in Table 1. Mean age was 45 (21–75) years. MPO levels ranged from 16 to 69 ng/ml (mean: 28.7 ng/ml), with no difference between men and
Fig. 1. Study design. FICF: free and informed consent form; RT: room temperature; CV: coefficient of variation; EDTA: ethylenediaminetetraacetic acid.
1652
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
Table 1 Characteristics of individuals participating in the study. Individuals (n = 40) Age, years Sex Males Females Individuals Controls With cardiac disease Medication AAS Statins Heparin Ticlopidine Angina Dyslipidemia Diabetes mellitus Arterial hypertension Renal insufficiency Inflammatory disease Neoplasm Myeloperoxidase (ng/ml)
44.7 (21–75) 17 (42.5%) 23 (57.5%) 25 (62.5%) 15 (37.5%) 11 (27.5%) 11 (27.5%) 6 (15%) 1 (2.5%) 4 (10%) 13 (32.5%) 3 (7.5%) 13 (32.5%) 4 (10%) 2 (5%) 1 (2.5%) 28.7 (16–69)
Age and myeloperoxidase: mean (minimum and maximum); AAS: acetyl salicylic acid.
women (31.0±12.4 vs. 27.0±12.2 ng/ml; P=0.31). MPO levels in the group of patients with cardiac disease were significantly higher than those in the group of healthy volunteers (35.0±15.4 vs. 24.9±8.2 ng/ml; P=0.03). The analytical intraassay and interassay CVs were 8.1% and 10.1%.
3.1. Effect of anticoagulants The comparison of MPO levels in different sample types revealed no significant differences between MPO-Hep (28.7±12.3 ng/ml) and serum collected in tubes with gel (28.9±13.4 ng/ml; P=0.87) or serum collected in tubes without gel (30.2±13.7 ng/ml; P=0.25). However, the results of samples collected in tubes containing EDTA for anticoagulation were significantly different from the results found for the MPO-Hep samples, with mean results about 1.8 times greater (51.4±15.5 vs. 28.7±12.3 ng/ml; Pb 0.001) (Fig. 2). The correlations between the results found for MPO-Gel and MPO-Serum and those for MPO-Hep were good (r=0.775 and r=0.792; Pb 0.001). There was no correlation between MPO-EDTA and MPO-Hep (r = 0.21; P = 0.20) (Fig. 3). The Bland–Altman plots [23] revealed that the mean differences of MPO-Hep from MPO-EDTA, MPO-Gel and MPO-Serum were 24.7 ng/ml (95%CI 19.5–30.0 ng/ml), 0.225 ng/ml (95%CI −2.54–2.99 ng/ml) and 1.55 ng/ml (95%CI −1.16–4.26 ng/ml) (Fig. 4).
Fig. 3. MPO levels in different sample types: correlation between MPO heparin and MPO-EDTA (a), MPO gel (b) and MPO serum (c). The dotted lines represent the line of equality at which Y = X.
3.2. Effect of storage conditions
Fig. 2. Effect of the different anticoagulants on MPO levels: comparison between plasma (with heparin and EDTA) and serum (with and without separator gel).
MPO levels for samples stored at different temperatures and for different periods of time are illustrated in Fig. 5. An increase in MPO levels in stored samples compared to the levels found in the baseline assay (36 h), (28.7±12.3 vs. 28.9±15.5 and 29.6± 15.2 ng/ml; PN 0.05, for baseline, 3 days at RT and refrigerator) being significant only from the 7day storage period, both for samples stored in the refrigerator (2–8 °C) and those stored at −20 °C (28.7 ±12.3 vs. 37.1±16.7 ng/ml; Pb 0.001 and 33.3±15.9 ng/ml; P=0.006) was observed. This increase remained constant for all other aliquots stored at all remaining time points. There were no differences between MPO levels of samples stored for 30, 90 or 180 days at −20 °C (37.9± 17.2 vs. 36.0 ±18.3 ng/ml; P =0.12 vs. 42.0 ±23.6 ng/ml; P= 0.21) or at −80 °C (36.7± 16.6 vs. 35.3 ± 19.0 ng/ml; P= 0.33 vs. 42.3±28.6 ng/ml; P=0.16). The analysis of sample stability over the entire storage time revealed a moderate
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
1653
Fig. 5. MPO levels assayed during storage. An increase in MPO levels was observed in stored aliquots along time, more markedly from 7 days onwards; increase then remained constant at later time points.
Fig. 4. Bland–Altman difference plots for MPO levels in different sample types: the solid line represents the mean difference between the reference sample (heparin) and EDTA (a), serum with (b) and without separator gel (c).
correlation between results (intraclass correlation coefficient [ICC]: 0.598; 95% CI 0.480–0.724; Pb 0.001). The comparison of samples stored at the same temperatures over time revealed better correlations: for those stored at −20 °C, ICC was 0.678 (95% CI 0.540–0.798; Pb 0.001), and for those stored at −80 °C, ICC was 0.667 (95% CI 0.507–0.795; Pb 0.001).
4. Discussion This study compared MPO results obtained from the analysis of heparinized plasma, which is the sample of choice according to the kit's manufacturer, with results obtained from plasma with EDTA and from serum collected in tubes with or without separator gel. No differences were found between MPO results of MPO-Hep and serum in tubes with or without separator gel. However, a significant difference was found in the MPO-EDTA results. Mean results of MPO-EDTA were about 107% greater than the results of MPO-Hep, and nine of the samples had results that were more than 150% greater.
Current knowledge suggests that MPO, a potential cause of progression and instability of atherosclerotic lesions during acute ischemic events, may be used as a marker of cardiovascular disease and provide information to improve patient diagnosis and prognosis [24]. However, little is known about the effect of preanalytical factors on MPO measurements. Studies have used different cut-off points because of differences in sample types and handling procedures. Therefore, the limits within which results can be used as the basis for decisions are uncertain, and further studies should evaluate analytical and preanalytical performances [22]. Several studies that evaluated other markers of cardiac disease, as well as the only two other studies about MPO published to date, found that preanalytical factors may affect measurements [25–35]. Chang et al. [31] compared MPO results from heparinized plasma and serum samples and found that serum results were considerably higher, although this could be attributed to the time the sample remained at room temperature for clot formation before centrifugation and separation of the material, and to the fact that the enzyme apparently continued to release from the leukocytes when whole blood was left standing at room temperature. No increases in MPO concentrations were found in samples place on ice bath after collection. Our study did not detect this difference: all our samples were put in an ice bath immediately after collection, and enzyme release was avoided. All tubes used to collect serum contained a clot activator. Samples collected in different tubes were found to have higher levels of MPO in serum and heparinized plasma than in plasma with EDTA or citrate [32]. In the heparinized plasma samples, MPO levels were about 10% higher than in EDTA plasma, and values for the serum samples were up to 100% higher. There was no significant difference between EDTA and citrate plasma in that study. Those findings differ from our results, in which values were considerably higher in EDTA samples. Some methodological and sample handling differences may explain the differences observed in the two studies. While in that study an automated chemiluminescent microparticle immunoassay was used, we used a manual method based on a sandwich ELISA and analyzed a larger number of samples. In our study samples were place on ice bath immediately after the collection. The changes observed in EDTA samples may be explained by the fact that when blood is collected in EDTA, neutrophils undergo morphological changes that are dependent on contact time and concentration, and minor changes may appear even at an optimal anticoagulant concentration. These changes include swelling of the neutrophils with loss of lobe structure, followed by loss of granulation and the appearance of vacuoles in the cytoplasm and in the nucleus of the cells. Three hours after blood collection, cells can disintegrate [36]. This loss of neutrophil granulation may be a contributing factor to the increase of MPO concentrations in plasma collected with EDTA. To avoid incorrect clinical
1654
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655
evaluations or risk stratification, blood collection in tubes with EDTA should be further investigated, and reference values should be determined. Similar discrepancies have been described for other markers. Roberts et al. [33] compared hs-CRP measurements in serum, heparinized plasma and EDTA plasma and found that, in spite of excellent correlations, EDTA plasma samples had lower levels. In contrast, another study found no significant differences between samples of serum, heparinized plasma and EDTA plasma collected simultaneously [34]. Our study also evaluated the stability of MPO according to different storage times and temperatures, from 3 days at room temperature (25 °C) or in a refrigerator (2–8 °C), to 6 months in a freezer (−20 °C and −80 °C). There was an increase in MPO levels from the first measurement to all others, and this increase was more marked from 7 days onwards. However, this difference does not seem to be a function of storage time or temperature because no systematic elevation of levels was found over time in the samples stored for longer periods (30, 90 and 180 days) at −20 °C or at −80 °C. Furthermore, aliquots to determine the analytical CVs also failed to show an increase in MPO levels at longer storage times. This difference, in agreement with the findings reported by Chang et al. [31], may be explained by the time that the plasma was in contact with cellular constituents while the samples remained at room temperature, which was approximately 2 h, from centrifugation to separation of the aliquots. In the study conducted by Chang et al. [31], the time during which the whole blood samples remained at room temperature before undergoing centrifugation had a significant effect on MPO levels in plasma; moreover, that increase was proportional to time, and the concentration was almost double when samples remained at room temperature for 1 h before centrifugation. No increases in MPO concentrations were found in samples put in an ice bath after collection, not even when centrifugation was performed at room temperature. In contrast, leukocytes apparently continued to release MPO in the samples stored at room temperature up to the time that they underwent centrifugation. Results reported by Shih et al. [32] also support this hypothesis. Plasma samples had a b10% difference in MPO levels after supernatant separation, storage at room temperature for 2 days, storage at 2–8 °C for 8 days and after 3 freeze–thaw cycles. However, the same study found a 4 times increase in the MPO levels of samples collected in tubes containing heparin and stored at room temperature for 2 h before centrifugation, whereas samples kept in an ice bath remained relatively stable. In contrast with the studies conducted by Chang et al. [31] and Shih et al. [32], who found that MPO levels increased in whole blood before sample centrifugation, we found that enzyme release seems to take place even after centrifugation. Therefore, even when samples are placed in an ice bath after collection, plasma must be separated immediately after centrifugation to avoid false increases in levels resulting from the continuous enzyme release into the plasma that is in contact with the cells. Our study results and those reported by Shih et al. [32] suggest that once plasma has been separated from the cellular constituents, MPO concentration is not apparently affected by storage temperature or time, which is useful information for investigators who intend to store samples for future measurements. What is of fundamental importance in MPO measurements and significantly affects the results is the time during which the sample, as whole blood or as plasma after centrifugation, remains in contact with cellular constituents. A common practice in clinical laboratories is to draw blood in serum-separator tubes because of the advantage of the barrier gel, which facilitates rapid separation of serum from cell constituents of blood and prevents hemolysis during prolonged storage. However, the measured concentrations of certain analytes may be reduced if specimens are stored in this type of tube because of adsorption by the
gel [35]. Our study did not show any significant differences between samples collected with heparin and serum with or without separator gel. Furthermore, despite some pair discrepancies, all of them within the expected total error for this analyte [37], there was a good correlation between MPO results for these 3 types of sample. This suggests that blood for the analysis of several cardiac markers may be collected in the same tube, which may shorten collection time, reduce costs and increase patient comfort. Some limitations to our study should be acknowledged. The fact that we did not know that the enzyme was likely to be released from the leukocytes into plasma after samples have been centrifuged, even when samples were put in an ice bath soon after collection, made our results difficult to interpret. Although they indicated the possibility that storage did not affect MPO levels, confirmation was only obtained from studies published subsequently. Studies in which samples remain in contact with cells before aliquot separation at room temperature and in an ice bath for predetermined times should be conducted to further explain the effect of this factor and define the rate at which MPO levels change. We evaluated the effect of storage on heparinized plasma samples only, and further studies should investigate such effect on other sample types. This study demonstrated that the collection of MPO in tubes containing EDTA alters in expressive way the results. Other aspects as time of storage and type of sample had no effect in the results. Further studies should be conducted to evaluate analytical and preanalytical variability. Measurement units should be standardized, populationbased reference ranges should be determined, and standard, automated and commercially available tests should be developed to shorten test time, facilitate use, and reduce analytical imprecision, which are the basic benchmarks to move this marker from the research environment to routine clinical practice.
References [1] Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation 2002;105:1135–43. [2] Hansson GK. Mechanisms of disease: inflammation, atherosclerosis and coronary artery disease. N Engl J Med 2005;352:1685–95. [3] Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler Thromb Vasc Biol 2005;25:1102–11. [4] Apple FS, Wu AHB, Mair J, Ravkilde J, Panteghini M, Tate J, et al. Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem 2005;51:810–24. [5] Naruko T, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, et al. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation 2002;106:2894–900. [6] Podrez EA, Schmitt D, Hoff HF, Hazen SL. Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro. J Clin Invest 1999;103:1547–60. [7] Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda SE, et al. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 2002;277:46116–22. [8] Nicholls S, Zheng L, Hazen SL. Formation of dysfunctional high-density lipoprotein by myeloperoxidase. Trends Cardiovasc Med 2005;15:212–9. [9] Hazen SL. Myeloperoxidase and plaque vulnerability. Arterioscler Thromb Vasc Biol 2004;24:1143–6. [10] Sugiyama S, Kugiyama K, Aikawa M, Nakamura S, Ogawa H, Libby P. Hypochlorous acid, a macrophage product, induces endothelial apoptosis and tissue factor expression. Involvement of myeloperoxidase-mediated oxidant in plaque erosion and thrombogenesis. Arterioscler Thromb Vasc Biol 2004;24:1309–14. [11] Vita JA, Brennan ML, Gokce N, Mann SA, Goormastic M, Shishehbor MH, et al. Serum myeloperoxidase levels independently predict endothelial dysfunction in humans. Circulation 2004;110:1134–9. [12] Baldus S, Heitzer T, Eiserich JP, Lau D, Mollnau H, Ortak M, et al. Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion. Free Radic Biol Med 2004;37:902–11. [13] Vasilyev N, Williams T, Brennan ML, Unzek S, Zhou X, Heinecke JW, et al. Myeloperoxidase-generated oxidants modulate left ventricular remodeling but not infarct size after myocardial infarction. Circulation 2005;112:2812–20. [14] Meuwese MC, Stroes WS, Hazen SL, van Miert JN, Kuivenhoven JA, Schaub RG, et al. Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals. J Am Coll Cardiol 2007;50:159–65. [15] Stefanescu A, Braun S, Ndrepepa G, Koppara T, Pavaci H, Mehilli J, et al. Prognostic value of plasma myeloperoxidase concentration in patients with stable coronary artery disease. Am Heart J 2008;155:356–60.
A.E. Wendland et al. / Clinica Chimica Acta 411 (2010) 1650–1655 [16] Baldus S, Heeschen C, Meinertz T, Zeiher AM, Eiserich JP, Münzel T, et al. Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes. Circulation 2003;108:1440–5. [17] Cavusoglu E, Ruwende C, Eng C, Chopra V, Yanamadala S, Clark LT, et al. Usefulness of baseline plasma myeloperoxidase levels as an independent predictor of myocardial infarction at two years in patients presenting with acute coronary syndrome. Am J Cardiol 2007;99:1364–8. [18] Mocatta TJ, Pilbrow AP, Cameron VA, Senthilmohan R, Frampton CM, Richards AM, et al. Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction. J Am Coll Cardiol 2007;49:1993–2000. [19] Brennan ML, Penn MS, Van Lente F, Nambi V, Shishehbor MH, Aviles RJ, et al. Prognostic value of myeloperoxidase in patients with chest pain. N Engl J Med 2003;349:1595–604. [20] Tang WHW, Brennan ML, Philip K, Tong W, Mann S, Van Lente F, et al. Plasma myeloperoxidase levels in patients with chronic heart failure. Am J Cardiol 2006;98: 796–9. [21] Tang WHW, Tong W, Troughton RW, Martin MG, Shrestha K, Borowski A, et al. Prognostic value and echocardiographic determinants of plasma myeloperoxidase levels in chronic heart failure. J Am Coll Cardiol 2007;49:2364–70. [22] Morrow DA. Appraisal of myeloperoxidase for evaluation of patients with suspected acute coronary syndromes. J Am Coll Cardiol 2007;49:2001–2. [23] Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;8:307–10. [24] Roman RM, Wendland AE, Polanczyk CA. Myeloperoxidase and coronary arterial disease: from research to clinical practice. Arq Bras Cardiol 2007;91:e12–9. [25] Stiegler H, Fischer Y, Jimenez JFV, Graf J, Filzmaier K, Fausten B, et al. Lower cardiac troponin T and I results in heparin-plasma than in serum. Clin Chem 2000;46: 1338–44. [26] Gerhardt W, Nordin G, Herbert AK, Burzell BL, Isaksson A, Gustavsson E, et al. Troponin T and I assays show decreased concentrations in heparin plasma compared
[27]
[28] [29] [30]
[31]
[32]
[33] [34] [35] [36] [37]
1655
with serum: lower recoveries in early than in late phases of myocardial injury. Clin Chem 2000;46:817–21. Ledue TB, Rifai N. Preanalytic and analytic sources of variation in C-reactive protein measurement: implications for cardiovascular disease risk assessment. Clin Chem 2003;49:1258–71. Lippi G, Salvagno GL, Montagnana M, Guidi GC. Measurement of Elecsys NTproBNP in serum, K2 EDTA and heparin plasma. Clin Biochem 2007;40:747–8. Shimizu H, Aono K, Masuta K, Asada H, Misaki A, Teraoka H. Stability of brain natriuretic peptide (BNP) in human blood samples. Clin Chim Acta 1999;285:169–72. Nowatzke WL, Cole TG. Stability of N-terminal pro-brain natriuretic peptide after storage frozen for one year and after multiple freeze–thaw cycles. Clin Chem 2003;49:1560–2. Chang PY, Wu TL, Hung CC, Tsao KC, Sun CF, Wu LL, et al. Development of an ELISA for myeloperoxidase on microplate: normal reference values and effect of temperature on specimen preparation. Clin Chim Acta 2006;373:158–63. Shih J, Datwyler SA, Hsu SC, Matias MS, Pacenti DP, Lueders C, et al. Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations. Clin Chem 2008;54:1076–9. Roberts WL, Schwarz EL, Ayanian S, Rifai N. Performance characteristics of a point of care C-reactive protein assay. Clin Chim Acta 2001;314:255–9. Rothkrantz-Kos S, Schmitz PJ, Bekers O, Menheere PPCA, van Dieijen-Visser MP. High-sensitivity C-reactive protein methods examined. Clin Chem 2002;48:359–62. Dasgupta A, Chow L, Tso G, Nazareno L. Stability of NT-proBNP in serum specimens collected in Becton Dickinson Vacutainer (SST) tubes. Clin Chem 2003;49:958–60. Narayanan S. The preanalytical phase — an important component of laboratory medicine. Am J Clin Pathol 2000;113:429–52. Biological Variation Database specifications — Westgard QC http://www.westgard. com/biodatabase1.htm.